發(fā)布時(shí)間：2018-01-04 00:15

  本文關(guān)鍵詞：攜Gelsolin單抗載紫杉醇靶向超聲造影劑的制備及體外顯影實(shí)驗(yàn)研究　出處：《大連醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 超聲造影劑 靶向 紫杉醇 凝溶膠蛋白

【摘要】：目的:以高分子聚合物聚乳酸-羥基乙酸(Poly lactic-co-glycolic acid,PLGA)為成膜材料,制備一種以腫瘤淋巴道轉(zhuǎn)移相關(guān)特異性膜抗原凝溶膠蛋白(Gelsolin,GSN)為靶點(diǎn)的高分子造影劑,并考察其體外尋靶及顯影能力。方法:通過(guò)改良的雙乳化法制備包裹紫杉醇(paclitaxel,PTX)的高分子PLGA-COOH造影劑,并以碳二亞胺法將載藥造影劑與Gelsolin單抗連接,制備出靶向載藥高分子超聲造影劑(PTX-PLGA-GSN)。利用光鏡及Malvern激光儀檢測(cè)此造影劑的分散度、粒徑、zeta電位等一般理化性質(zhì);取適量該靶向載藥造影劑以甲醇溶解萃取包載的紫杉醇,以高效液相色譜儀分析其包封率、載藥量及體外釋藥特性;采用免疫熒光法及流式細(xì)胞儀檢測(cè)Gelsolin單抗與造影劑表面的連接情況;體外培養(yǎng)小鼠肝癌細(xì)胞系具備不同淋巴道轉(zhuǎn)移的能力的Hca-F和Hca-P細(xì)胞,設(shè)立4組實(shí)驗(yàn)組,Hca-F細(xì)胞非靶向PLGA組、Hca-F細(xì)胞抗體封閉組、Hca-P細(xì)胞GSN-PLGA組、Hca-F細(xì)胞GSN-PLGA組,每組加入相應(yīng)的香豆素-6標(biāo)記的造影劑處理后,于激光共聚焦顯微鏡下觀察每組細(xì)胞對(duì)造影劑的攝取情況,流式細(xì)胞儀檢測(cè)各組細(xì)胞的熒光強(qiáng)度,評(píng)價(jià)靶向造影劑的體外尋靶能力;稱取一定量造影劑重懸于雙蒸水中,調(diào)整濃度分別為1mg/ml、0.5mg/ml、0.25mg/ml,并以脫氣水為對(duì)照組,于超聲診斷儀造影模式下觀察體外顯影情況。結(jié)果:PTX-PLGA-GSN納米超聲造影劑的平均粒徑為(328.59±3.82)nm,zeta電位為(-11.46±1.19)mV,包封率及載藥量分別為(83.1±2.12)%、(8.31±0.21)%,藥物體外釋放實(shí)驗(yàn)顯示30d紫杉醇緩釋率約為90.63%。流式細(xì)胞術(shù)測(cè)得PTX-PLGA-GSN表面GSN單抗連接率高達(dá)98.31%。體外攝取實(shí)驗(yàn)顯示小鼠腹水型肝癌高淋巴道轉(zhuǎn)移細(xì)胞株(Hca-F)可攝取較多的攜Gelsolin單抗PLGA造影劑,流式細(xì)胞儀檢測(cè)Hca-F細(xì)胞GSN-PLGA組細(xì)胞熒光強(qiáng)度平均為(189.09±1.14),明顯高于Hca-P細(xì)胞GSN-PLGA組的(140.15±0.98),差異具有統(tǒng)計(jì)學(xué)意義(P0.05),Hca-F細(xì)胞非靶向PLGA組及Hca-F細(xì)胞抗體封閉組的細(xì)胞熒光強(qiáng)度分別為(108.39±1.29)、(123.31±1.25),亦顯著低于Hca-F細(xì)胞GSN-PLGA組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。體外顯影實(shí)驗(yàn)顯示造影劑回聲均勻細(xì)膩,后方回聲無(wú)衰減,成像效果較好,且隨著造影劑濃度的增加,造影效果更優(yōu)。結(jié)論:成功制備出攜Gelsolin單抗載紫杉醇靶向超聲造影劑且包封率及載藥量均較高,且緩釋性能較好,體外尋靶實(shí)驗(yàn)顯示此造影劑的靶向性較強(qiáng),體外顯影效果亦較好。
[Abstract]:Objective: to polymer poly lactic acid glycolic acid (Poly lactic-co-glycolic, acid, PLGA) as membrane material, a preparation for lymphatic metastasis of tumor associated antigen specific membrane gelsolin (Gelsolin, GSN) as the polymer contrast agent targeting, and investigate its in vitro targeting and developing ability. By parcel of paclitaxel by double emulsification method improved (paclitaxel, PTX) PLGA-COOH polymer contrast agent, and the carbon two imide method drug loaded contrast agent with Gelsolin mAb connection, prepared targeting drug loaded polymer ultrasound contrast agent (PTX-PLGA-GSN). By using optical microscope and Malvern laser dispersion detection the agent particle size, zeta potential of the general physical and chemical properties; apply the drug targeting contrast agent dissolved in methanol extraction entrapped paclitaxel, with HPLC analysis of the encapsulation rate, drug loading and release characteristics in vitro by immunofluorescence; The optical method and flow cytometry Gelsolin monoclonal antibody and contrast agent surface connection; cultured mouse hepatocellular carcinoma cell lines with different lymph node metastasis in Hca-F and Hca-P cells, the establishment of the 4 experimental groups, Hca-F cell non targeting PLGA group, Hca-F cell antibody group, Hca-P GSN-PLGA cells group, Hca-F cells GSN-PLGA group, each group with the corresponding contrast agent of coumarin labeled -6, the laser scanning confocal microscope on the uptake of contrast agents in each cell, the fluorescence intensity of cells were detected by flow cytometry, evaluation of targeted contrast agent targeting; weigh a certain amount of resuspended in double contrast agent the water vapor, adjust the concentration were 1mg/ml, 0.5mg/ml, 0.25mg/ml, and the degassed water as control group, to observe the in vitro development in ultrasonic diagnosis instrument contrast mode. Results: the average PTX-PLGA-GSN nano ultrasound contrast agent Particle size (328.59 + 3.82) nm, zeta potential (-11.46 + 1.19) mV, the entrapment efficiency and drug loading were (83.1 + 2.12)% and (8.31 + 0.21)%, in vitro drug release experiments showed that 30d paclitaxel release rate of about 90.63%. were measured by flow cytometry PTX-PLGA-GSN surface GSN mAb connection rate is as high as 98.31%. uptake in vitro experiments showed that mouse hepatocarcinoma cell lines with high lymphatic metastasis (Hca-F) can absorb more with Gelsolin monoclonal antibody PLGA contrast agent, flow cytometry Hca-F GSN-PLGA cells fluorescence intensity average (189.09 + 1.14), was significantly higher in Hca-P cells group (GSN-PLGA 140.15 + 0.98), the difference was statistically significant (P0.05), Hca-F cell non targeting PLGA and Hca-F cell antibody group the cell fluorescence intensity were (108.39 + 1.29), (123.31 + 1.25), was significantly lower than that of Hca-F cells in GSN-PLGA group, the difference was statistically significant (P0.05) in vitro development. Experiments show that the contrast agent echo uniform fine, rear echo attenuation, good imaging results, and with the increasing concentration of contrast, contrast is better. Conclusion: we successfully synthesized with paclitaxel loaded Gelsolin monoclonal antibody targeted ultrasound contrast agent and the encapsulation efficiency and drug loading were higher, and the sustained release performance in vitro the target experiment showed that targeting the strong contrast agent, the in vitro development effect is also good.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R445.1

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